Chromogranin A (CgA) is a protein that was identified and isolated in 1965 from chromaffin cells of bovine adrenal medulla (Banks et al. 1965. Biochem J 97: 40C-1C; Taupenot et al. 2010. New Eng J Med. 348: 1134-49). Chromaffin cells are neuroendocrine cells found mostly in the medulla of the adrenal glands in mammals. Chromogranin A is an established tumor marker in a variety of neuroendocrine tumors, a heterogeneous group of rare neoplasms from neuroendocrine cells comprising multiple endocrine neoplasia, type 1 and type 2 (MEN1/MEN2), medullary thyroid carcinoma, carcinoid tumors, islet cell tumors, pheochromocytoma/paraganglioma, poorly differentiated/small cell/atypical lung carcinoid, small cell carcinoma of the lung, Merkel cell carcinoma (Deftos et al. 1991. Endocr. Rev. 12: 181-7; Corti et al. 1996. Br J Cancer 73: 924-32) and mentioned in several guidelines (Ramage et al. 2012. Gut 61: 6-32; Vinik et al. 2010. NANETS Pancreas 39(6): 713-734; Pape et al. 2012. ENETS 95: 135-156).
Human Chromogranin A has a sequence of 439 amino acid residues (see SEQ ID NO:1) constituting a 49 kDa acidic glycoprotein that is stored and released from the chromaffin granules of endocrine cells, neurons and neurendocrines cells along with their respective hormones, neurotransmitters, and neuropeptides (Kim et al. 2001. Cell 106: 499-509).
Chromogranin A is the main member of the chromogranin/secretogranin family which consists of a group of proteins derived from different genes but share a number of characteristics, namely an abundance of acidic amino acid residues and numerous pairs of basic amino acids as potential positions for post-translational processing (Metz-Boutigue et al. 1993. Eur. J. Biochem 217: 247-257) and cleavage.
CgA is the precursor of several biologically active peptide fragments that have been described in human and in other species: vasostatins (Drees et al. 1991. Endocrinology 129: 3381-7), chromostatin (Galindo et al. 1991. Proc Natl Acad Sci USA 88: 1426-30), chromacins (Strub et al. 1997. J Biol Chem 272: 11928-36), pancreastatin (Tatemoto et al. 1986. Nature 324: 476-8), WE-14 (Curry et al. 1992. FEBS Lett 301: 319-321), catestatin (Mahata et al. 1997. J Clin Invest 100: 1623-33), parastatin (Fasciotto et al. 1993. Endocrinology 133: 461-6) and GE-25 (Kirchmair et al. 1995. Biochem J 310 (Pt 1): 331-6). In the UniProt database, additional peptides for human Chromogranin A (accession number: P 10645) are mentioned, based on the cleavage sites that can predict the peptides to be released: vasostatin-1 comprises amino acid sequence 1-76, vasostatin-2 comprises amino acid sequence 1-113, EA-92 comprises amino acid sequence 116-207, ES-43 comprises amino acid sequence 210-242, pancreastatin comprises amino acid sequence 254-301, SS-18 comprises amino acid sequence 304-321, WE-14 comprises amino acid sequence 324-337, WA-8 comprises amino acid sequence 324-331, LF-19 comprises amino acid sequence 340-358, AL-11 comprises amino acid sequence 362-372, GV-19 comprises amino acid sequence 375-393, GR-44 comprises amino acid sequence 395-438 and ER-37 comprises amino acid sequence 402-438. Moreover, it has been shown that different neurendocrine cells can process the molecule differently (Portela-Gomes et al. 2001. J Histochem Cytochem 4: 483-90).
Chromgranin A has been described as a biomarker for a number of diseases and conditions including cancer, for example prostate cancer (WO 2013/070088 A1; WO 2013/070089 A1; U.S. Pat. No. 6,238,877 B1; WO 2012/065025 A2).
Today, four non radiative CE-marked commercial assays for the detection of Chromogranin A are available: The Cis-Bio ELISA assay (Cisbio Bioassays, Codolet, France) uses two monoclonal antibodies directed against epitopes corresponding to amino acids 145-197 and 219-234, the DAKO ELISA assay (Dako Denmark A/S, Glostrup, Denmark) uses rabbit polyclonal antibodies directed against a 23 kDa C-terminal fragment, the Euro-Diagnostica NEOLISA™ sandwich ELISA assay (Euro Diagnostica AB, Malmö, Sweden) uses two monoclonal antibodies directed against epitopes corresponding to amino acids 236-251 and 264-279 (also see WO 2011/135035 A1 and WO 99/58980 A1).
The only available fully automated assay for the detection of Chromogranin A is the Chromogranin A KRYPTOR assay (Thermo Fisher Scientific B.R.A.H.M.S GmbH, Hennigsdorf, Germany) which uses two monoclonal antibodies, one monoclonal antibody binding to an epitope corresponding to amino acids 250-301 (Popovici et al. 2014. Clin Biochem 47: 87-91).
Due to high proteolysis of the molecule, the measured concentration in these assays can vary depending on the storage of the collected sample over the time and depending on the fragment being measured by the antibodies in the assay, thus an improved assay for Chromogranin A should address the most stable fragments of the molecule and be assessed in terms of sample stability under different storage conditions.
Different publications describe the impact of antibody epitopes and assay design on clinical performance of Chromogranin A immunoassays (Corti et al. 1996. Eur J. Biochem 235: 275-280, Stridsberg et al. 2003. J Endocrinol 177: 337-41).